HMGB1在人脐血造血干细胞归巢和增殖分化中的作用及其机制研究
发布时间：2019-04-20 13:56
【摘要】： 中文摘要 背景与目的高迁移率族蛋白B1(high mobility group box 1,HMGB1)是一种DNA结合蛋白,组织损伤后释放至胞外,招募多种类型干细胞,参与受损组织修复。造血干细胞移植(haematopoietic stem cell transplantation ,HSCT)的预处理必然导致骨髓组织受损,释放HMGB1,推测其可能参与造血重建。本实验将首次观察预处理后骨髓基质细胞HMGB1的释放、HMGB1对人脐血造血干细胞归巢和增殖分化的影响,并对其机制予以探讨;以期为促进HSCT术后造血重建寻找新靶点。方法体外培养人骨髓基质细胞,加速器照射后,利用ELISA方法检测培养上清中HMGB1含量的变化。利用MACS系统分选人脐血CD34+细胞,HMGB1与其共培养6天,通过流式细胞术检测脐血CD34+细胞分化指标(CD13、CD14、CD11c、CD41,CD71)的变化。利用克隆形成实验观察HMGB1与对造血干细胞增殖分化的影响。应用transwell小室趋化装置观察HMGB1对人脐血CD34+细胞的迁移活性的影响。流式细胞术检测HMGB1的受体RAGE、TLR2和TLR4在人脐血CD34+细胞上的表达。利用抗-RAGE抗体、抗- TLR2抗体和抗- TLR4抗体阻断RAGE、TLR2和TLR4,重复趋化实验,体外观察RAGE、TLR2和TLR4是否参与HMGB1可能诱导的人脐血CD34+细胞迁移。结果1.经X线照射后骨髓基质细胞培养上清中HMGB1含量增高:人骨髓基质细胞,加速器照射(12Gy)后,培养上清中HMGB1含量为(4.3±0.9)ng/ml,较不照射组HMGB1含量(0.4±0.2)ng/ml升高(p0.01)。2.HMGB1表面受体检测人脐血CD34+细胞表达HMGB1的受体RAGE(43.1±7.2)%、TLR2(36.1±6.6)%和TLR4(23.1±5.2)%。3.HMGB1促进CD34+细胞向红系和粒单系增殖分化:脐血分选富集的纯度为(98.25±0.93)%,与HMGB1体外液体共培养6天后,和对照组比较,红系(CD71)和粒单系(CD13、CD14、CD11c)标记的表达明显增强,分别为CD13(18.4±3.8 vs 32.6±5.9)%、CD14(12.6±2.7 vs 25.4±4.4)%、CD11c(9.8±2.1 vs 20.3±3.9)%、、CD71(26.6±4.6 vs 47.1±7.4)%,而巨核系标记CD41(1.1±0.4% vs 1.3±0.5%)的表达无明显变化。同样,克隆形成实验示共培养14天后,红系集落、粒-巨噬细胞集落和总集落的生成较对照组明显增多(p0.05)。4.HMGB1诱导脐血CD34+细胞的迁移:HMGB1在一定的浓度范围内随浓度递增其对人脐血CD34+细胞的迁移作用逐渐增强,当HMGB1浓度为100 ng/ml时,趋化活性最强,趋化指数为3.96±0.46,与对照组比较差异显著(p0.01),抗-RAGE抗体可部分抑制HMGB1对脐血CD34+细胞的迁移作用。 结论照射后骨髓基质细胞培养上清HMGB1含量明显升高;HMGB1可促进人脐血CD34+细胞向粒单核及红系分化,并促进红系集落和粒-巨噬细胞集落的生成;一定浓度的HMGB1可加强脐血CD34+细胞迁移功能,此作用有可能通过RAGE介导。
[Abstract]:Background and objective High mobility group protein B1 (high mobility group box 1 (HMGB1) is a DNA binding protein that is released into extracellular cells after tissue damage and enlists many types of stem cells to participate in the repair of damaged tissues. The pretreatment of hematopoietic stem cell transplantation (haematopoietic stem cell transplantation, HSCT) inevitably results in bone marrow tissue damage, and the release of HMGB1, speculates that it may be involved in hematopoietic reconstitution. In this study, the release of HMGB1 from bone marrow stromal cells after pretreatment and the effect of HMGB1 on homing, proliferation and differentiation of human umbilical cord blood hematopoietic stem cells were observed for the first time, and the mechanism was discussed in order to find a new target for promoting hematopoiesis reconstruction after HSCT. Methods Human bone marrow stromal cells (BMSCs) were cultured in vitro. After irradiation with accelerator, the content of HMGB1 in culture supernatant was detected by ELISA. Human umbilical cord blood CD34 cells were selected by MACS system and co-cultured with HMGB1 for 6 days. The changes of differentiation index (CD13,CD14,CD11c,CD41,CD71) of CD34 cells in cord blood were detected by flow cytometry. The effects of HMGB1 on the proliferation and differentiation of hematopoietic stem cells were observed by clone formation assay. Transwell chamber chemotactic assay was used to observe the effect of HMGB1 on the migration activity of human umbilical cord blood CD34 cells. Flow cytometry was used to detect the expression of HMGB1 receptor RAGE,TLR2 and TLR4 in human umbilical cord blood CD34 cells. Using anti-RAGE antibody, anti-TLR2 antibody and anti-TLR4 antibody to block the repeated chemotaxis test of RAGE,TLR2 and TLR4, the effects of RAGE,TLR2 and TLR4 on CD34 cell migration induced by HMGB1 were observed in vitro. Outcome 1. The content of HMGB1 in the culture supernatant of bone marrow stromal cells was increased after X-ray irradiation. After 12Gy, the content of HMGB1 in the culture supernatant was (4.3 卤0.9) ng/ml,. 2. The expression of HMGB1 receptor RAGE in human umbilical cord blood CD34 cells was detected by HMGB1 surface receptor (43.1 卤7.2)%, and the HMGB1 content was (0.40.2) ng/ml higher than that of non-irradiated group (p0.01.HMGB1 surface receptor was (43.1 卤7.2)%). TLR2 (36.1 卤6.6)% and TLR4 (23.1 卤5.2)%. 3. HMGB1 promoted the proliferation and differentiation of CD34 cells into erythroid and granulocytic lines. The purity of cord blood separation and enrichment was (98.25 卤0.93)%. After 6 days of co-culture with HMGB1, HMGB1 was co-cultured with HMGB1 for 6 days. Compared with the control group, the expression of CD71 and CD13,CD14,CD11c markers were significantly increased, which were (18.4 卤3.8 vs 32.6 卤5.9)% for CD13 and (12.6 卤2.7 vs 25.4 卤4.4)% for CD14, respectively. The expression of CD11c (9.8 卤2.1 vs 20.3 卤3.9)%, CD71 (26.6 卤4.6 vs 47.1 卤7.4)%, and megakaryocyte marker CD41 (1.1 卤0.4% vs 1.3 卤0.5%) did not change significantly. Similarly, clone formation tests showed that after 14 days of co-culture, the erythroid colonies, 4.HMGB1 induced the migration of cord blood CD34 cells: the migration of HMGB1 to human umbilical cord blood CD34 cells increased gradually with the increasing concentration of HMGB1 within a certain range of concentration. 4. HMGB1 induced the migration of human umbilical cord blood CD34 cells in a certain concentration range. 4. HMGB1 induced the migration of human umbilical cord blood CD34 cells in a certain concentration range (p0.05). When the concentration of HMGB1 was 100 ng/ml, the chemotactic activity was the strongest, and the chemotactic index was 3.96 卤0.46, which was significantly different from that of the control group (p0.01). Anti-RAGE antibody could partially inhibit the migration of HMGB1 to CD34 cells in cord blood. Conclusion HMGB1 can promote the differentiation of human umbilical cord blood CD34 cells into granulocyte monocytes and erythroid cells, and promote the formation of erythroid colony and granulocyte-macrophage colony, and increase the content of HMGB1 in the culture supernatant of bone marrow stromal cells after irradiation, and HMGB1 can promote the differentiation of human cord blood CD34 cells into granulocyte monocytes and erythrocytes. A certain concentration of HMGB1 can enhance the migration of cord blood CD34 cells, which may be mediated by RAGE.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
本文编号：2461661
[Abstract]:Background and objective High mobility group protein B1 (high mobility group box 1 (HMGB1) is a DNA binding protein that is released into extracellular cells after tissue damage and enlists many types of stem cells to participate in the repair of damaged tissues. The pretreatment of hematopoietic stem cell transplantation (haematopoietic stem cell transplantation, HSCT) inevitably results in bone marrow tissue damage, and the release of HMGB1, speculates that it may be involved in hematopoietic reconstitution. In this study, the release of HMGB1 from bone marrow stromal cells after pretreatment and the effect of HMGB1 on homing, proliferation and differentiation of human umbilical cord blood hematopoietic stem cells were observed for the first time, and the mechanism was discussed in order to find a new target for promoting hematopoiesis reconstruction after HSCT. Methods Human bone marrow stromal cells (BMSCs) were cultured in vitro. After irradiation with accelerator, the content of HMGB1 in culture supernatant was detected by ELISA. Human umbilical cord blood CD34 cells were selected by MACS system and co-cultured with HMGB1 for 6 days. The changes of differentiation index (CD13,CD14,CD11c,CD41,CD71) of CD34 cells in cord blood were detected by flow cytometry. The effects of HMGB1 on the proliferation and differentiation of hematopoietic stem cells were observed by clone formation assay. Transwell chamber chemotactic assay was used to observe the effect of HMGB1 on the migration activity of human umbilical cord blood CD34 cells. Flow cytometry was used to detect the expression of HMGB1 receptor RAGE,TLR2 and TLR4 in human umbilical cord blood CD34 cells. Using anti-RAGE antibody, anti-TLR2 antibody and anti-TLR4 antibody to block the repeated chemotaxis test of RAGE,TLR2 and TLR4, the effects of RAGE,TLR2 and TLR4 on CD34 cell migration induced by HMGB1 were observed in vitro. Outcome 1. The content of HMGB1 in the culture supernatant of bone marrow stromal cells was increased after X-ray irradiation. After 12Gy, the content of HMGB1 in the culture supernatant was (4.3 卤0.9) ng/ml,. 2. The expression of HMGB1 receptor RAGE in human umbilical cord blood CD34 cells was detected by HMGB1 surface receptor (43.1 卤7.2)%, and the HMGB1 content was (0.40.2) ng/ml higher than that of non-irradiated group (p0.01.HMGB1 surface receptor was (43.1 卤7.2)%). TLR2 (36.1 卤6.6)% and TLR4 (23.1 卤5.2)%. 3. HMGB1 promoted the proliferation and differentiation of CD34 cells into erythroid and granulocytic lines. The purity of cord blood separation and enrichment was (98.25 卤0.93)%. After 6 days of co-culture with HMGB1, HMGB1 was co-cultured with HMGB1 for 6 days. Compared with the control group, the expression of CD71 and CD13,CD14,CD11c markers were significantly increased, which were (18.4 卤3.8 vs 32.6 卤5.9)% for CD13 and (12.6 卤2.7 vs 25.4 卤4.4)% for CD14, respectively. The expression of CD11c (9.8 卤2.1 vs 20.3 卤3.9)%, CD71 (26.6 卤4.6 vs 47.1 卤7.4)%, and megakaryocyte marker CD41 (1.1 卤0.4% vs 1.3 卤0.5%) did not change significantly. Similarly, clone formation tests showed that after 14 days of co-culture, the erythroid colonies, 4.HMGB1 induced the migration of cord blood CD34 cells: the migration of HMGB1 to human umbilical cord blood CD34 cells increased gradually with the increasing concentration of HMGB1 within a certain range of concentration. 4. HMGB1 induced the migration of human umbilical cord blood CD34 cells in a certain concentration range. 4. HMGB1 induced the migration of human umbilical cord blood CD34 cells in a certain concentration range (p0.05). When the concentration of HMGB1 was 100 ng/ml, the chemotactic activity was the strongest, and the chemotactic index was 3.96 卤0.46, which was significantly different from that of the control group (p0.01). Anti-RAGE antibody could partially inhibit the migration of HMGB1 to CD34 cells in cord blood. Conclusion HMGB1 can promote the differentiation of human umbilical cord blood CD34 cells into granulocyte monocytes and erythroid cells, and promote the formation of erythroid colony and granulocyte-macrophage colony, and increase the content of HMGB1 in the culture supernatant of bone marrow stromal cells after irradiation, and HMGB1 can promote the differentiation of human cord blood CD34 cells into granulocyte monocytes and erythrocytes. A certain concentration of HMGB1 can enhance the migration of cord blood CD34 cells, which may be mediated by RAGE.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329
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相关期刊论文 前2条
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2 郝一文,李亚明,刘显智;辐照的血管内皮对造血干细胞迁徙的影响[J];中华放射医学与防护杂志;2005年05期
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